Susarla K. Shankar

A draft map of the human proteome

The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides does not exist yet. Here we present a draft map of the human proteome using high-resolution Fourier-transform mass spectrometry. In-depth proteomic profiling of 30 histologically normal human samples, including 17 adult tissues, 7 fetal tissues and 6 purified primary haematopoietic cells, resulted in identification of proteins encoded by 17,294 genes accounting for approximately 84% of the total annotated protein-coding genes in humans. A unique and comprehensive strategy for proteogenomic analysis enabled us to discover a number of novel protein-coding regions, which includes translated pseudogenes, non-coding RNAs and upstream open reading frames. This large human proteome catalogue (available as an interactive web-based resource at http://www.humanproteomemap.org) will complement available human genome and transcriptome data to accelerate biomedical research in health and disease.

Aluminium in Alzheimer's disease: are we still at a crossroad?

Abstract.Aluminium, an environmentally abundant non-redox trivalent cation has long been implicated in the pathogenesis of Alzheimer’s disease (AD). However, the definite mechanism of aluminium toxicity in AD is not known. Evidence suggests that trace metal homeostasis plays a crucial role in the normal functioning of the brain, and any disturbance in it can exacerbate events associated with AD. The present paper reviews the scientific literature linking aluminium with AD. The focus is on aluminium levels in brain, region-specific and subcellular distribution, its relation to neurofibrillary tangles, amyloid beta, and other metals. A detailed mechanism of the role of aluminium in oxidative stress and cell death is highlighted. The importance of complex speciation chemistry of aluminium in relation to biology has been emphasized. The debatable role of aluminium in AD and the cross-talk between aluminium and genetic susceptibility are also discussed. Finally, it is concluded based on extensive literature that the neurotoxic effects of aluminium are beyond any doubt, and aluminium as a factor in AD cannot be discarded. However, whether aluminium is a sole factor in AD and whether it is a factor in all AD cases still needs to be understood.

Tat Protein of Human Immunodeficiency Virus Type 1 Subtype C Strains Is a Defective Chemokine

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD) is correlated with increased monocyte migration to the brain, and the incidence of HAD among otherwise asymptomatic subjects appears to be lower in India than in the United States and Europe (1 to 2% versus 15 to 30%). Because of the genetic differences between HIV-1 strains circulating in these regions, we sought to identify viral determinants associated with this difference. We targeted Tat protein for these studies in view of its association with monocyte chemotactic function. Analyses of Tat sequences representing nine subtypes revealed that at least six amino acid residues are differentially conserved in subtype C Tat (C-Tat). Of these, cysteine (at position 31) was highly (>99%) conserved in non-subtype C viruses and more than 90% of subtype C viruses encoded a serine. We hypothesized a compromised chemotactic function of C-Tat due to the disruption of CC motif and tested it with the wild type C-Tat (CS) and its two isogenic variants (CC and SC) derived by site-directed mutagenesis. We found that the CS natural variant was defective for monocyte chemotactic activity without a loss in the transactivation property. While the CC mutant is functionally competent for both the functions, in contrast, the SC mutant was defective in both. Therefore, the loss of the C-Tat chemotactic property may underlie the reduced incidence of HAD; although not presenting conclusive evidence, this study provides the first evidence for a potential epidemiologic phenomenon associated with biological differences in the subtype C viruses.

NMDA Receptor Activation by HIV-Tat Protein Is Clade Dependent

In countries infected with HIV clade B, some patients develop a rapidly progressive dementia that if untreated results in death. In regions of the world infected with HIV clade C, only milder forms of cognitive impairment have been recognized. HIV-infected macrophages are the principal mediators of dementia. HIV clade C, however, efficiently infects macrophages and HIV-infected macrophages are found in the brains of clade C-infected patients. HIV-infected macrophages release Tat protein, which may act directly on neurons to cause toxicity. We found that Tat released from Tat-expressing cells was at least 1000-fold more toxic than recombinant Tat protein. We determined whether Tat could interact with NMDA receptors and whether these interactions are clade dependent. It is demonstrated that Tat binds directly to the NMDA receptor leading to excitotoxicity. The Cys 30-Cys 31 motif in Tat is critical for exciting the NMDA receptor and the Cys31Ser mutation found in clade C Tat has a significantly attenuated neurotoxic response. Through molecular modeling and site-directed mutagenesis, we predict that Cys 31 disrupts the disulfide bond between Cys 744 and Cys 798 on the NR1 subunit of the NMDA receptor by directly interacting with Cys 744 leading to a free thiol group on Cys 798 and subsequent persistent activation of the NMDA receptor.

Thiol Oxidation and Loss of Mitochondrial Complex I Precede Excitatory Amino Acid-Mediated Neurodegeneration

Human ingestion of “chickling peas” from the plantLathyrus sativus, which contains an excitatory amino acid, l-BOAA (l-β-N-oxalylamino-l-alanine), leads to a progressive corticospinal neurodegenerative disorder, neurolathyrism. Exposure to l-BOAA, but not its optical enantiomer d-BOAA, causes mitochondrial dysfunction as evidenced by loss of complex I activity in vitro in male mouse brain slices and in vivo in selected regions of mouse CNS (lumbosacral cord and motor cortex). Loss of complex I activity in lumbosacral cord after l-BOAA administration to mice was accompanied by concurrent loss of glutathione. The inhibited complex I activity in mitochondria isolated from lumbosacral cord of animals treated with l-BOAA rebounded after incubation with the thiol-reducing agent dithiothreitol, indicating that oxidation of protein thiols to disulfides was responsible for enzyme inhibition. The inhibition of complex I could be abolished by pretreatment with antioxidant thiols such as glutathione ester and α-lipoic acid. Chronic treatment of male mice, but not female mice, withl-BOAA resulted in loss of complex I activity and vacuolation and dendritic swelling of neurons in the motor cortex and lumbar cord, paralleling the regionality of the aforementioned biochemical effects on CNS mitochondria. These results support the view that thiol oxidation and concomitant mitochondrial dysfunction (also implicated in other neurodegenerative disorders), occurring downstream of glutamate receptor activation by l-BOAA, are primary events leading to neurodegeneration. Maintenance of protein thiol homeostasis by thiol delivery agents could potentially offer protection against excitotoxic insults such as those seen withl-BOAA.

Induction of brain cytochrome P-450IIE1 by chronic ethanol treatmen

Cytochrome P-450 mediated metabolism is potentially involved in the expression of the pharmacological and/or toxicological effects of a wide variety of drugs and environmental chemicals upon tissues which contain this metabolic system. In the present investigation, the presence of cytochrome P-450IIE1 and associated mono-oxygenase activities in brain and the effect of chronic ethanol treatment on brain cytochrome P-450 (P-450) were studied. Aniline hydroxylase, N-nitroso-dimethylamine N-demethylase and p-nitrophenol hydroxylase activities (known to be mediated by P-450IIE1) were detectable in brain microsomes from untreated rats and were about 5%, 125% and 8.3%, respectively, of the corresponding hepatic levels. Chronic ethanol treatment resulted in induction of the above enzyme activities in brain microsomes by 243%, 496% and 155%, respectively. Intake of ethanol for a prolonged period also resulted in the induction of total P-450 in the brain (150% of the control). Addition of the antisera raised against rat liver cytochrome P-450IIE1 markedly inhibited brain microsomal p-nitrophenol hydroxylase activity. Immunoblot analysis of rat brain microsomes using the above antisera also revealed the induction of brain cytochrome P-450IIE1 following chronic ethanol administration. Immunocytochemical localization of cytochrome P-450IIE1 using the above antisera, revealed the preferential localization of the enzyme in the neuronal cell bodies in the cortex, hippocampus, basal ganglia, hypothalamic nuclei and reticular nuclei in the brainstem of rats treated chronically with ethanol. Based upon these studies, it is conceivable that chronic alcohol ingestion could enhance the sensitivity of certain regions of the brain to environmental chemicals that are metabolized to more toxic derivatives by the P-450 system.

Xenobiotic metabolism in human brain ― presence of cytochrome P-450 and associated mono-oxygenases

The cytochromes P-450, a family of heme proteins, play an important role in the oxidation of drugs and carcinogens, as well as endogenous substrates. We report the presence of cytochrome P-450 and associated mono-oxygenase activity in human brain regions and their selective enrichment in the brainstem. Immunocytochemical studies on human medulla with antibodies raised to phenobarbital-inducible rat liver cytochrome P-450 indicate that the enzyme is primarily localized in the neuronal cell bodies and to a lesser extent in the axons. These observations indicate that the human brain could be involved in metabolism of xenobiotics and endogenous compounds, mediated through cytochrome P-450.

Rat brain cytochromes P-450: catalytic, immunochemical properties and inducibility of multiple forms

Cytochrome P-450 (P-450) and associated mono-oxygenase activities were estimated in male and female rat brain microsomes. The P-450 concentration in male rat brain was one-tenth the corresponding hepatic levels, which is considerably higher than earlier reports. A distinct sex-related difference was observed in the levels of total P-450 and mono-oxygenase activities known to be mediated by P-450b,e; the female brain levels were 60% of those in the males. Immunoinhibition and immunoblot studies using antisera to P-450b,e and P-450c,d indicated the presence of multiple forms of P-450, immunologically similar to P-450b,e, P-450c and P-450d in the rat brain. Prior treatment with phenobarbital resulted in two-fold increase of total P-450 and selective induction of aminopyrine N-demethylase (APD) and morphine N-demethylase (MND) activities. Administration of 3-methylcholanthrene, selectively induced the levels of ethoxycoumarin O-deethylase (ECD) and arylhydrocarbon hydroxylase, although the levels of total P-450 were not increased. 3-Methylcholanthrene induction was also accompanied by a shift in the absorption maximum of the reduced carbon monoxide difference spectrum from 452 to 448 nm. Immunocytochemical localization using antibodies to P-450b,e indicated the presence of P-450 predominantly in the neuronal cell bodies and to a lesser extent in the fibre tracts in cerebral cortex, cerebellum, thalamus, hypothalamus, hippocampus and brainstem. These studies indicate that the brain contains significant amounts of P-450, which exists in multiple forms and can be selectively induced by prior exposure to phenobarbital or 3-methylcholanthrene.

Identification of Subtype C Human Immunodeficiency Virus Type 1 by Subtype-Specific PCR and Its Use in the Characterization of Viruses Circulating in the Southern Parts of India

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) subtype C viruses are associated with nearly half of worldwide HIV-1 infections and are most predominant in India and the southern and eastern parts of Africa. Earlier reports from India identified the preponderance of subtype C and a small proportion of subtype A viruses. Subsequent reports identifying multiple subtypes suggest new introductions and/or their detection due to extended screening. The southern parts of India constitute emerging areas of the epidemic, but it is not known whether HIV-1 infection in these areas is associated with subtype C viruses or is due to the potential new introduction of non-subtype C viruses. Here, we describe the development of a specific and sensitive PCR-based strategy to identify subtype C-viruses (C-PCR). The strategy is based on amplifying a region encompassing a long terminal repeat and gag in the first round, followed by two sets of nested primers; one amplifies multiple subtypes, while the other is specific to subtype C. The common HIV and subtype C-specific fragments are distinguishable by length differences in agarose gels and by the difference in the numbers of NF-κB sites encoded in the subtype C-specific fragment. We implemented this method to screen 256 HIV-1-infected individuals from 35 towns and cities in four states in the south and a city in the east. With the exception of single samples of subtypes A and B and a B/C recombinant, we found all to be infected with subtype C viruses, and the subtype assignments were confirmed in a subset by using heteroduplex mobility assays and phylogenetic analysis of sequences. We propose the use of C-PCR to facilitate rapid molecular epidemiologic characterization to aid vaccine and therapeutic strategies.

Japanese encephalitis virus antigen in the human brain and its topographic distribution

This study reports the pathological findings and the distribution of viral antigen in the brains of 13 confirmed and autopsied cases of Japanese encephalitis (JE) in correlation with other virus-specific immunological parameters measured in the cerebrospinal fluid (CSF) antemortem. Japanese encephalitis virus (JEV)-specific antibodies were detected in the CSF of 10 of 13 patients, JEV antigen was detected in the CSF of 7 of 13 and JEV-specific immune complexes were detected in the CSF of 3 of 11 patients. Viral antigen was localised immunocytochemically in the brain tissue of 11 of 13 cases, indicating, that viral antigen could not be cleared from the tissues by the antibody. The topographic distribution of the tissue-associated antigen in the thalamus, hippocampus, substantia nigra and medulla oblongata explain the evolution of post JE sequelae.